Suppressing PD-L1 Expression via AURKA Kinase Inhibition Enhances Natural Killer Cell-Mediated Cytotoxicity against Glioblastoma.

Immunotherapies have shown significant promise as an impactful strategy in cancer treatment. However, in glioblastoma multiforme (GBM), the most prevalent primary brain tumor in adults, these therapies have demonstrated lower efficacy than initially anticipated. Consequently, there is an urgent need for strategies to enhance the effectiveness of immune treatments. AURKA has been identified as a potential drug target for GBM treatment. An analysis of the GBM cell transcriptome following AURKA inhibition revealed a potential influence on the immune system. Our research revealed that AURKA influenced PD-L1 levels in various GBM model systems in vitro and in vivo. Disrupting AURKA function genetically led to reduced PD-L1 levels and increased MHC-I expression in both established and patient-derived xenograft GBM cultures. This process involved both transcriptional and non-transcriptional pathways, partly implicating GSK3ß. Interfering with AURKA also enhanced NK-cell-mediated elimination of GBM by reducing PD-L1 expression, as evidenced in rescue experiments. Furthermore, using a mouse model that mimics GBM with patient-derived cells demonstrated that Alisertib decreased PD-L1 expression in living organisms. Combination therapy involving anti-PD-1 treatment and Alisertib significantly prolonged overall survival compared to vehicle treatment. These findings suggest that targeting AURKA could have therapeutic implications for modulating the immune environment within GBM cells.

Revolutionizing Brain Tumor Care: Emerging Technologies and Strategies.

Glioblastoma multiforme (GBM) is one of the most aggressive forms of brain tumor, characterized by a daunting prognosis with a life expectancy hovering around 12-16 months. Despite a century of relentless research, only a select few drugs have received approval for brain tumor treatment, largely due to the formidable barrier posed by the blood-brain barrier. The current standard of care involves a multifaceted approach combining surgery, irradiation, and chemotherapy. However, recurrence often occurs within months despite these interventions. The formidable challenges of drug delivery to the brain and overcoming therapeutic resistance have become focal points in the treatment of brain tumors and are deemed essential to overcoming tumor recurrence. In recent years, a promising wave of advanced treatments has emerged, offering a glimpse of hope to overcome the limitations of existing therapies. This review aims to highlight cutting-edge technologies in the current and ongoing stages of development, providing patients with valuable insights to guide their choices in brain tumor treatment.

Stimulation of ectopically expressed muscarinic receptors induces IFN-γ but suppresses IL-2 production by inhibiting activation of pAKT pathways in primary T cells.

T cell antigen receptor stimulation induces tyrosine phosphorylation of downstream signaling molecules and the phosphatidylinositol, Ras, MAPK, and PI3 kinase pathways, leading to T cell activation. Previously, we reported that the G-protein-coupled human muscarinic receptor could bypass tyrosine kinases to activate the phosphatidylinositol pathway and induce interleukin-2 production in Jurkat leukemic T cells. Here, we demonstrate that stimulating G-protein-coupled muscarinic receptors (M1 and synthetic hM3Dq) can activate primary mouse T cells if PLCß1 is coexpressed. Resting peripheral hM3Dq+PLCß1 (hM3Dq/ß1) T cells did not respond to clozapine, an hM3Dq agonist, unless they were preactivated by TCR and CD28 stimulation which increased hM3Dq and PLCß1 expression. This permitted large calcium and phosphorylated ERK responses to clozapine. Clozapine treatment induced high IFN-γ, CD69, and CD25 expression, but surprisingly did not induce substantial IL-2 in hM3Dq/ß1 T cells. Importantly, costimulation of both muscarinic receptors plus the TCR even led to reduced IL-2 expression, suggesting a selective inhibitory effect of muscarinic receptor costimulation. Stimulation of muscarinic receptors induced strong nuclear translocation of NFAT and NFκB and activated AP-1. However, stimulation of hM3Dq led to reduced IL-2 mRNA stability which correlated with an effect on the IL-2 3'UTR activity. Interestingly, stimulation of hM3Dq resulted in reduced pAKT and its downstream pathway. This may explain the inhibitory impact on IL-2 production in hM3Dq/ß1T cells. Moreover, an inhibitor of PI3K reduced IL-2 production in TCR-stimulated hM3Dq/ß1 CD4 T cells, suggesting that activating the pAKT pathway is critical for IL-2 production in T cells.

Dietary restriction of cysteine and methionine sensitizes gliomas to ferroptosis and induces alterations in energetic metabolism.

Ferroptosis is mediated by lipid peroxidation of phospholipids containing polyunsaturated fatty acyl moieties. Glutathione, the key cellular antioxidant capable of inhibiting lipid peroxidation via the activity of the enzyme glutathione peroxidase 4 (GPX-4), is generated directly from the sulfur-containing amino acid cysteine, and indirectly from methionine via the transsulfuration pathway. Herein we show that cysteine and methionine deprivation (CMD) can synergize with the GPX4 inhibitor RSL3 to increase ferroptotic cell death and lipid peroxidation in both murine and human glioma cell lines and in ex vivo organotypic slice cultures. We also show that a cysteine-depleted, methionine-restricted diet can improve therapeutic response to RSL3 and prolong survival in a syngeneic orthotopic murine glioma model. Finally, this CMD diet leads to profound in vivo metabolomic, proteomic and lipidomic alterations, highlighting the potential for improving the efficacy of ferroptotic therapies in glioma treatment with a non-invasive dietary modification.

A cell state specific metabolic vulnerability to GPX4-dependent ferroptosis in glioblastoma.

Glioma cells hijack developmental transcriptional programs to control cell state. During neural development, lineage trajectories rely on specialized metabolic pathways. However, the link between tumor cell state and metabolic programs is poorly understood in glioma. Here we uncover a glioma cell state-specific metabolic liability that can be leveraged therapeutically. To model cell state diversity, we generated genetically engineered murine gliomas, induced by deletion of p53 alone (p53) or with constitutively active Notch signaling (N1IC), a pathway critical in controlling cellular fate. N1IC tumors harbored quiescent astrocyte-like transformed cell states while p53 tumors were predominantly comprised of proliferating progenitor-like cell states. N1IC cells exhibit distinct metabolic alterations, with mitochondrial uncoupling and increased ROS production rendering them more sensitive to inhibition of the lipid hydroperoxidase GPX4 and induction of ferroptosis. Importantly, treating patient-derived organotypic slices with a GPX4 inhibitor induced selective depletion of quiescent astrocyte-like glioma cell populations with similar metabolic profiles.

Therapeutic Drug-Induced Metabolic Reprogramming in Glioblastoma.

Glioblastoma WHO IV (GBM), the most common primary brain tumor in adults, is a heterogenous malignancy that displays a reprogrammed metabolism with various fuel sources at its disposal. Tumor cells primarily appear to consume glucose to entertain their anabolic and catabolic metabolism. While less effective for energy production, aerobic glycolysis (Warburg effect) is an effective means to drive biosynthesis of critical molecules required for relentless growth and resistance to cell death. Targeting the Warburg effect may be an effective venue for cancer treatment. However, past and recent evidence highlight that this approach may be limited in scope because GBM cells possess metabolic plasticity that allows them to harness other substrates, which include but are not limited to, fatty acids, amino acids, lactate, and acetate. Here, we review recent key findings in the literature that highlight that GBM cells substantially reprogram their metabolism upon therapy. These studies suggest that blocking glycolysis will yield a concomitant reactivation of oxidative energy pathways and most dominantly beta-oxidation of fatty acids.

A systemic cell stress signal confers neuronal resilience toward oxidative stress in a Hedgehog-dependent manner.

Cells possess several conserved adaptive mechanisms to respond to stress. Stress signaling is initiated to reestablish cellular homeostasis, but its effects on the tissue or systemic levels are far less understood. We report that the secreted luminal domain of the endoplasmic reticulum (ER) stress transducer CREB3L2 (which we name TAILS [transmissible activator of increased cell livability under stress]) is an endogenous, cell non-autonomous activator of neuronal resilience. In response to oxidative insults, neurons secrete TAILS, which potentiates hedgehog signaling through direct interaction with Sonic hedgehog (SHH) and its receptor PTCH1, leading to improved antioxidant signaling and mitochondrial function in neighboring neurons. In an in vivo model of ischemic brain injury, administration of TAILS enables survival of CNS neurons and fully preserves cognitive function in behavioral tests. Our findings reveal an SHH-mediated, cell non-autonomous branch of cellular stress signaling that confers resilience to oxidative stress in the mature brain, providing protection from ischemic neurodegeneration.

Dissecting the treatment-naive ecosystem of human melanoma brain metastasis.

Melanoma brain metastasis (MBM) frequently occurs in patients with advanced melanoma; yet, our understanding of the underlying salient biology is rudimentary. Here, we performed single-cell/nucleus RNA-seq in 22 treatment-naive MBMs and 10 extracranial melanoma metastases (ECMs) and matched spatial single-cell transcriptomics and T cell receptor (TCR)-seq. Cancer cells from MBM were more chromosomally unstable, adopted a neuronal-like cell state, and enriched for spatially variably expressed metabolic pathways. Key observations were validated in independent patient cohorts, patient-derived MBM/ECM xenograft models, RNA/ATAC-seq, proteomics, and multiplexed imaging. Integrated spatial analyses revealed distinct geography of putative cancer immune evasion and evidence for more abundant intra-tumoral B to plasma cell differentiation in lymphoid aggregates in MBM. MBM harbored larger fractions of monocyte-derived macrophages and dysfunctional TOX+CD8+ T cells with distinct expression of immune checkpoints. This work provides comprehensive insights into MBM biology and serves as a foundational resource for further discovery and therapeutic exploration.

Induction of Synthetic Lethality by Activation of Mitochondrial ClpP and Inhibition of HDAC1/2 in Glioblastoma.

PURPOSE: Novel therapeutic targets are critical to unravel for the most common primary brain tumor in adults, glioblastoma (GBM). We have identified a novel synthetic lethal interaction between ClpP activation and HDAC1/2 inhibition that converges on GBM energy metabolism. EXPERIMENTAL DESIGN: Transcriptome, metabolite, and U-13C-glucose tracing analyses were utilized in patient-derived xenograft (PDX) models of GBM. Orthotopic GBM models were used for in vivo studies. RESULTS: We showed that activation of the mitochondrial ClpP protease by mutant ClpP (Y118A) or through utilization of second-generation imipridone compounds (ONC206 and ONC212) in combination with genetic interference of HDAC1 and HDAC2 as well as with global (panobinostat) or selective (romidepsin) HDAC inhibitors caused synergistic reduction of viability in GBM model systems, which was mediated by interference with tricarboxylic acid cycle activity and GBM cell respiration. This effect was partially mediated by activation of apoptosis along with activation of caspases regulated chiefly by Bcl-xL and Mcl-1. Knockdown of the ClpP protease or ectopic expression of a ClpP D190A mutant substantially rescued from the inhibition of oxidative energy metabolism as well as from the reduction of cellular viability by ClpP activators and the combination treatment, respectively. Finally, utilizing GBM PDX models, we demonstrated that the combination treatment of HDAC inhibitors and imipridones prolonged host survival more potently than single treatments or vehicle in vivo. CONCLUSIONS: Collectively, these observations suggest that the efficacy of HDAC inhibitors might be significantly enhanced through ClpP activators in model systems of human GBM.

Methodological Approaches for Assessing Metabolomic Changes in Glioblastomas.

Glioblastoma (GBM), a highly malignant primary brain tumor, inevitably leads to death. In the last decade, a variety of novel molecular characteristics of GBMs were unraveled. The identification of the mutation in the IDH1 and less commonly IDH2 gene was surprising and ever since has nurtured research in the field of GBM metabolism. While initially thought that mutated IDH1 were to act as a loss of function mutation it became clear that it conferred the production of an oncometabolite that in turn substantially reprograms GBM metabolism. While mutated IDH1 represents truly the tip of the iceberg, there are numerous other related observations in GBM that are of significant interest to the field, including the notion that oxidative metabolism appears to play a more critical role than believed earlier. Metabolic zoning is another important hallmark of GBM since it was found that the infiltrative margin that drives GBM progression reveals enrichment of fatty acid derivatives. Consistently, fatty acid metabolism appears to be a novel therapeutic target for GBM. How metabolism in GBM intersects is another pivotal issue that appears to be important for its progression and response and resistance to therapies. In this review, we will summarize some of the most relevant findings related to GBM metabolism and cell death and how these observations are influencing the field. We will provide current approaches that are applied in the field to measure metabolomic changes in GBM models, including the detection of unlabeled and labeled metabolites as well as extracellular flux analysis.

ZAP70, too little, too much can lead to autoimmunity.

Establishing both central and peripheral tolerance requires the appropriate TCR signaling strength to discriminate self- from agonist-peptide bound to self MHC molecules. ZAP70, a cytoplasmic tyrosine kinase, directly interacts with the TCR complex and plays a central and requisite role in TCR signaling in both thymocytes and peripheral T cells. By studying ZAP70 hypomorphic mutations in mice and humans with a spectrum of hypoactive or hyperactive activities, we have gained insights into mechanisms of central and peripheral tolerance. Interestingly, both hypoactive and hyperactive ZAP70 can lead to the development of autoimmune diseases, albeit through distinct mechanisms. Immature thymocytes and mature T cells rely on normal ZAP70 function to complete their development in the thymus and to modulate T cell responses in the periphery. Hypoactive ZAP70 function compromises key developmental checkpoints required to establish central tolerance, allowing thymocytes with potentially self-reactive TCRs a greater chance to escape negative selection. Such 'forbidden clones' may escape into the periphery and may pose a greater risk for autoimmune disease development since they may not engage negative regulatory mechanisms as effectively. Hyperactive ZAP70 enhances thymic negative selection but some thymocytes will, nonetheless, escape negative selection and have greater sensitivity to weak and self-ligands. Such cells must be controlled by mechanisms involved in anergy, expansion of Tregs, and upregulation of inhibitory receptors or signaling molecules. However, such potentially autoreactive cells may still be able to escape control by peripheral negative regulatory constraints. Consistent with findings in Zap70 mutants, the signaling defects in at least one ZAP70 substrate, LAT, can also lead to autoimmune disease. By dissecting the similarities and differences among mouse models of patient disease or mutations in ZAP70 that affect TCR signaling strength, we have gained insights into how perturbed ZAP70 function can lead to autoimmunity. Because of our work and that of others on ZAP70, it is likely that perturbations in other molecules affecting TCR signaling strength will be identified that also overcome tolerance mechanisms and cause autoimmunity. Delineating these molecular pathways could lead to the development of much needed new therapeutic targets in these complex diseases.

Aurora kinase A inhibition reverses the Warburg effect and elicits unique metabolic vulnerabilities in glioblastoma.

Aurora kinase A (AURKA) has emerged as a drug target for glioblastoma (GBM). However, resistance to therapy remains a critical issue. By integration of transcriptome, chromatin immunoprecipitation sequencing (CHIP-seq), Assay for Transposase-Accessible Chromatin sequencing (ATAC-seq), proteomic and metabolite screening followed by carbon tracing and extracellular flux analyses we show that genetic and pharmacological AURKA inhibition elicits metabolic reprogramming mediated by inhibition of MYC targets and concomitant activation of Peroxisome Proliferator Activated Receptor Alpha (PPARA) signaling. While glycolysis is suppressed by AURKA inhibition, we note an increase in the oxygen consumption rate fueled by enhanced fatty acid oxidation (FAO), which was accompanied by an increase of Peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC1α). Combining AURKA inhibitors with inhibitors of FAO extends overall survival in orthotopic GBM PDX models. Taken together, these data suggest that simultaneous targeting of oxidative metabolism and AURKAi might be a potential novel therapy against recalcitrant malignancies.

Targeting super-enhancers reprograms glioblastoma central carbon metabolism.

The concept that tumor cells demand a distinct form of metabolism was appreciated almost a century ago when the German biochemist Otto Warburg realized that tumor cells heavily utilize glucose and produce lactic acid while relatively reducing oxidative metabolism. How this phenomenon is orchestrated and regulated is only partially understood and seems to involve certain transcription factors, including c-Myc, HIF1A and others. The epigenome eintails the posttranslational modification of histone proteins which in turn are involved in regulation of transcription. Recently, it was found that cis-regulatory elements appear to facilitate the Warburg effects since several genes encoding for glycolysis and associated pathways are surrounded by enhancer/super-enhancer regions. Disruption of these regions by FDA-approved HDAC inhibitors suppressed the transcription of these genes and elicited a reversal of the Warburg effect with activation of transcription factors facilitating oxidative energy metabolism with increases in transcription factors that are part of the PPARA family. Therefore, combined targeting of HDACs and oxidative metabolism suppressed tumor growth in patient-derived xenograft models of solid tumors, including glioblastoma.

RAB38 Facilitates Energy Metabolism and Counteracts Cell Death in Glioblastoma Cells.

Glioblastoma is a high-grade glial neoplasm with a patient survival of 12-18 months. Therefore, the identification of novel therapeutic targets is an urgent need. RAB38 is a GTPase protein implicated in regulating cell proliferation and survival in tumors. The role of RAB38 in glioblastoma is relatively unexplored. Here, we test the hypothesis that RAB38 regulates glioblastoma growth using human glioblastoma cell lines. We found that genetic interference of RAB38 resulted in a decrease in glioblastoma growth through inhibition of proliferation and cell death induction. Transcriptome analysis showed that RAB38 silencing leads to changes in genes related to mitochondrial metabolism and intrinsic apoptosis (e.g., Bcl-xL). Consistently, rescue experiments demonstrated that loss of RAB38 causes a reduction in glioblastoma viability through downregulation of Bcl-xL. Moreover, RAB38 knockdown inhibited both glycolysis and oxidative phosphorylation. Interference with RAB38 enhanced cell death induced by BH3-mimetics. RAB38 antagonists are under development, but not yet clinically available. We found that FDA-approved statins caused a rapid reduction in RAB38 protein levels, increased cell death, and phenocopied some of the molecular changes elicited by loss of RAB38. In summary, our findings suggest that RAB38 is a potential therapeutic target for glioblastoma treatment.

Cell-Penetrating CEBPB and CEBPD Leucine Zipper Decoys as Broadly Acting Anti-Cancer Agents.

Transcription factors are key players underlying cancer formation, growth, survival, metastasis and treatment resistance, yet few drugs exist to directly target them. Here, we characterized the in vitro and in vivo anti-cancer efficacy of novel synthetic cell-penetrating peptides (Bpep and Dpep) designed to interfere with the formation of active leucine-zipper-based dimers by CEBPB and CEBPD, transcription factors implicated in multiple malignancies. Both peptides similarly promoted apoptosis of multiple tumor lines of varying origins, without such effects on non-transformed cells. Combined with other treatments (radiation, Taxol, chloroquine, doxorubicin), the peptides acted additively to synergistically and were fully active on Taxol-resistant cells. The peptides suppressed expression of known direct CEBPB/CEBPD targets IL6, IL8 and asparagine synthetase (ASNS), supporting their inhibition of transcriptional activation. Mechanisms by which the peptides trigger apoptosis included depletion of pro-survival survivin and a required elevation of pro-apoptotic BMF. Bpep and Dpep significantly slowed tumor growth in mouse models without evident side effects. Dpep significantly prolonged survival in xenograft models. These findings indicate the efficacy and potential of Bpep and Dpep as novel agents to treat a variety of cancers as mono- or combination therapies.

Telomere biology disorders.

Telomere biology disorders (TBD) are a heterogeneous group of diseases arising from germline mutations affecting genes involved in telomere maintenance. Telomeres are DNA-protein structures at chromosome ends that maintain chromosome stability; their length affects cell replicative potential and senescence. A constellation of bone marrow failure, pulmonary fibrosis, liver cirrhosis and premature greying is suggestive, however incomplete penetrance results in highly variable manifestations, with idiopathic pulmonary fibrosis as the most common presentation. Currently, the true extent of TBD burden is unknown as there is no established diagnostic criteria and the disorder often is unrecognised and underdiagnosed. There is no gold standard for measuring telomere length and not all TBD-related mutations have been identified. There is no specific cure and the only treatment is organ transplantation, which has poor outcomes. This review summarises the current literature and discusses gaps in understanding and areas of need in managing TBD.

Cbl-b deficiency prevents functional but not phenotypic T cell anergy.

T cell anergy is an important peripheral tolerance mechanism. We studied how T cell anergy is established using an anergy model in which the Zap70 hypermorphic mutant W131A is coexpressed with the OTII TCR transgene (W131AOTII). Anergy was established in the periphery, not in the thymus. Contrary to enriched tolerance gene signatures and impaired TCR signaling in mature peripheral CD4 T cells, CD4SP thymocytes exhibited normal TCR signaling in W131AOTII mice. Importantly, the maintenance of T cell anergy in W131AOTII mice required antigen presentation via MHC-II. We investigated the functional importance of the inhibitory receptor PD-1 and the E3 ubiquitin ligases Cbl-b and Grail in this model. Deletion of each did not affect expression of phenotypic markers of anergic T cells or T reg numbers. However, deletion of Cbl-b, but not Grail or PD-1, in W131AOTII mice restored T cell responsiveness and signaling. Thus, Cbl-b plays an essential role in the establishment and/or maintenance of unresponsiveness in T cell anergy.

Effects of Fermentation Conditions Using Lactobacillus plantarum on the Charantin, Stigmasterol Glucoside and ß-sitosterol Glucoside Contents of Bitter Gourd (Momordica charantia L.) Juice.

Bitter gourd fruits contain high amounts of charantin, stigmasterol glucoside and ß-sitosterol glucoside, which have been shown to provide health benefits for humans. However, the bitterness of the fruit means they are rarely consumed. This study aimed to assess the effects of Lactobacillus plantarum fermentation, which has previously been reported to effectively reduce bitterness, on the contents of these compounds. The current results suggest that Lactobacillus plantarum fermentation should be considered as a potential approach to enhance the levels of these compounds in bitter gourd juice.

Epigenetic Targeting of Mcl-1 Is Synthetically Lethal with Bcl-xL/Bcl-2 Inhibition in Model Systems of Glioblastoma.

Apoptotic resistance remains a hallmark of glioblastoma (GBM), the most common primary brain tumor in adults, and a better understanding of this process may result in more efficient treatments. By utilizing chromatin immunoprecipitation with next-generation sequencing (CHIP-seq), we discovered that GBMs harbor a super enhancer around the Mcl-1 locus, a gene that has been known to confer cell death resistance in GBM. We utilized THZ1, a known super-enhancer blocker, and BH3-mimetics, including ABT263, WEHI-539, and ABT199. Combined treatment with BH3-mimetics and THZ1 led to synergistic growth reduction in GBM models. Reduction in cellular viability was accompanied by significant cell death induction with features of apoptosis, including disruption of mitochondrial membrane potential followed by activation of caspases. Mechanistically, THZ1 elicited a profound disruption of the Mcl-1 enhancer region, leading to a sustained suppression of Mcl-1 transcript and protein levels, respectively. Mechanism experiments suggest involvement of Mcl-1 in the cell death elicited by the combination treatment. Finally, the combination treatment of ABT263 and THZ1 resulted in enhanced growth reduction of tumors without induction of detectable toxicity in two patient-derived xenograft models of GBM in vivo. Taken together, these findings suggest that combined epigenetic targeting of Mcl-1 along with Bcl-2/Bcl-xL is potentially therapeutically feasible.

SIRT7 deficiency suppresses inflammation, induces EndoMT, and increases vascular permeability in primary pulmonary endothelial cells.

Acute lung injury (ALI), a common condition in critically ill patients, has limited treatments and high mortality. Aging is a risk factor for ALI. Sirtuins (SIRTs), central regulators of the aging process, decrease during normal aging and in aging-related diseases. We recently showed decreased SIRT7 expression in lung tissues and fibroblasts from patients with pulmonary fibrosis compared to controls. To gain insight into aging-related mechanisms in ALI, we investigated the effects of SIRT7 depletion on lipopolysaccharide (LPS)-induced inflammatory responses and endothelial barrier permeability in human primary pulmonary endothelial cells. Silencing SIRT7 in pulmonary artery or microvascular endothelial cells attenuated LPS-induced increases in ICAM1, VCAM1, IL8, and IL6 and induced endomesenchymal transition (EndoMT) with decreases in VE-Cadherin and PECAM1 and increases in collagen, alpha-smooth muscle actin, TGFß receptor 1, and the transcription factor Snail. Loss of endothelial adhesion molecules was accompanied by increased F-actin stress fibers and increased endothelial barrier permeability. Together, these results show that an aging phenotype induced by SIRT7 deficiency promotes EndoMT with impaired inflammatory responses and dysfunction of the lung vascular barrier.